| IFN(interferon,IFN) is a kind of anti-virus, regulating cell growth and differentiation, immune-modulated protein. It also is first-discovered, most-studied, first-cloned and first-applied to clinical therapy cytokine. In veterinary medicine, it is important to prevent disease and cure virus-disease, to study vaccine adjuvant et al.In this test, three pairs primmer were respectively designed in according to public IFN CDS sequences (poIFN-α: M 28623; poIFN-βS 41178; poIFN-γ: X 53085) in GenBank. Applying RT-PCR (reverse transcription-polymerase chain reaction, RT-PCR), three segments of sequences (poIFN-α:570bp; poIFN-β: 561bp; poIFN-γ: 501bp) were cloned from cDNA of landrace spleen cell induced by conA. By T/A clone principles, three segments of sequences were linked with pMD-T18 vector, and those linked vectors were transformed into E.coli. DH5α. The positive clones were verified from PCR with bacterium and plasmid template, or from digesting with restrictive enzyme. The conclusions of sequence determined showed that poIFNs were cloned. Analyzed aminophenol sequence, there were seven mutation sides (38,40,43,86,98,112,118 side) in poIFN-αprotein; there were only one mutation side( 22 side E to G) in poIFN-βprotein; there were only one mutation side(143 side K to N).The poLFNs were respectively subcloned into pET-28(a) (EcoR I and HindⅢ) and pGEX-KG (BamH I and HindⅢ). The positive clones were validated by PCR, enzyme digesting and sequence determining. The poIFNs were expressed in E.coli. BL21 with pET-28(a) and pGEX-KG after induced by IPTG respectively. The high effciency expression of fusion proteins was obtained by detected with SDS-PAGE. The proteins (pET-28(a)-poIFNs) were mixed with fushi adjuvant, and the admixtures were injected into New Zealand white rabbit. The high effective antibody levels were obtained, and the indirect ELISA titers were respectively rabbit-anti-poIFN-α: 10×255; rabbit-anti-polFN-β: 10×26; rabbit-anti-poIFN-γ: 10×25. Rabbit IgG were deposited by saturated ammonium sulfate, then IgG were purified with A-50.The proteins(pET-28(a)-poIFN-α,β,γ) were mixed with fushi adjuvant, and the admixtures were injected into Balb/c rice. The hybridoma were generated by fusion of SP2/0 myeloma cells and the splenocytes from immunized mice with PEG4000. The positive clones were screened by proteins ofpGEX-KG-IFN-α,β,γand negative control with lysate of BL21(pET-28(a)). The five hybridoma cell lines were screened and polFN-αhad been developed one line(2H12), polFN-βhad been developed two lines(1G8, 3A1), polFN-γhad been developed two lines(3D5, 4B4). The results of chromosome counts showed that all hybridoma cells were fusion of SP2/0 myeloma cells and the splenocytes. The monoclonal antibody specificities were verified with indirect ELISA and western-blotting. The Mab isotypes were detected with Mab isotypes Kits and the results were respectively of 2H12: IgG1; 3A1: lgG2a; 1G8:IgG2b; 3D5: IgG1; 4B4: IgG2a. The ELISA titers of the ascite fluids induced by the five hybridomas were respectively of 2H12: 100×26; 3A1: 100×28; 1G8: 100×27; 3D5: 100×28; 4B4: 100×26.
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